Highlights Lecture #21 Spring 2017

Highlights Translation 3

1. Translation is a target for action of antibiotics. This works because the phenomenon of translation in prokaryotes is different enough from that of eukaryotes that specific inhibitors of prokaryotic translation can be found that have no effect on eukaryotic translation. As a result, prokaryotes can be killed by some antibiotics without any effect on eukaryotes. One translational inhibitor of prokaryotic translation is the antibiotic puromycin, which acts to prematurely terminate prokaryotic translation at the elongation phase. This happens because puromycin looks like a tRNA and fits into the A (or P) site of the ribosome. In the A site, it can be attached to the growing polypeptid chain, but it is soon released, causing premature termination. In addition, puromycin can bind to the P site of ribosomes and prevents anything from binding there.

2. Eukaryotic mRNAs have a 5′ cap and a 3′ polyA tail. Eukaryotic translation occurs much like prokaryotic translation, but the factors are differently named. One important difference of eukaryotic translation is that the initiation complex in eukaryotes involves both the 5′ cap and the 3′ polyA sequence in a looped structure. The 5′ cap and 3′ poly-A sequence both play roles in helping to increase the stability of eukaryotic mRNAs.

3. Post-translational (= after translation has occurred) processing of proteins gives them their final structure and properties. One such modification was discussed for insulin. This involves proteolytic cleavage at two places in the insulin precursor, giving rise to a final structure that has two chains held together by disulfide bonds.

4. Proteins do not have long lifetimes in the cell. They are broken down in cells in cellular structures called proteasomes. Proteins targeted for degradation are tagged with the peptide known as ubiquitin.

5. Selenocysteine is a rare amino acid occasionally incorporated into proteins. It is sometimes called the 21st amino acid. It is the only modified amino acid in proteins that gets there by direct incorporation rather than post-translational modification. It is because of this amino acid that trace amounts of selenium are needed in the diet.

6. Proper folding of proteins is important. A complex in E. coli that facilitates proper folding of proteins is the GroES/GroEL complex, which provides a chamber for a protein to fold in without interference of other proteins during the folding process.

Biotechnology

1. Biotechnology employs knowledge of the processes of DNA replication, transcription, and translation to make useful products.

2. The modern era of biotechnology began with the discovery of restriction endonucleases (=restriction enzymes). These are enzymes isolated from bacterial cells that bind to DNA, recognize a specific DNA sequence, and cut at that sequence.

3. Many restriction enzymes recognize specific inverted repeat sequences and cut there. For EcoRI, the cut DNA fragments have overhanging ends called “sticky” that can be used to form base pairs that are useful for putting pieces back together with DNA ligase.

4. Restriction enzymes are bacterial defense systems. In normal bacterial cells, they are paired with an enzyme called a methylase. The function of the methylase is to put a methyl group on the sequence the restriction enzyme would otherwise cut. Cellular DNA is protected in this way. Invading virus sequences, however, aren’t methylated, so their DNAs get cut by the restriction enzymes.

5. If one piece of a DNA molecule is linked to a foreign DNA molecule, a recombinant molecule is created. If one uses DNA ligase to link a DNA to a circular DNA that replicates in bacteria (called a plasmid), then the foreign DNA will be replicated in the bacterial cells. If the plasmid has a promoter that can control transcription, the plasmid is known as an expression vector. An expression vector provides a means of using bacteria to transcribe and translate foreign genes.

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